The primary objective of the project's second year is the precise identification of the promoters and regulatory regions controlling expression of the metBJLF genes. Mapping of promoter sites will be accomplished with the electron microscope through binding of RNA polymerase holoenzyme to the genomes of select lambda met transducing phage which carry all or part of the metBJLF gene cluster. RNA polymerase binding sites will be correlated with dA/dT rich regions of DNA sequence by partial denaturation mapping of the same phage genomes. The transcriptional organization of the met genes will be analyzed by RNA/DNA filter hybridization assays which use as probes the separated DNA strands of met transducing phage. These assays will also be used to quantitate endogenous metBJLF mRNA levels in wild type E. coli K12 cultures and in cultures of mutant strains containing genetic defects affecting regulation of expression of the met genes. Preliminary cloning experiments using down sized DNA fragments isolated from transposon mutagenized, restriction endonuclease digested transducing phage chromosomes are to be continued. High and low copy number plasmid vectors will be used to evaluate gene dosage effects associated with these DNA fragments. Detailed restriction endonuclease maps of these DNA fragments will also be accomplished. In addition, transposon mutagenesis and deletion analysis of characterized transducing phage chromosomes will continue.